REVISED ABSTRACT Limb abnormalities caused by disruption of the human short-stature homeobox (SHOX) gene and its surrounding regions are quite common, affecting up to 1:1000 individuals. Deficiencies in SHOX gene function cause shortening of the zeugopod, and are associated with several short-stature syndromes including Lri-Weill dyschondrosteosis (LWD) and Langer syndrome. Importantly, more than one-third of patients with LWD do not have mutations within the SHOX gene, but have deletions that remove noncoding sequences up and downstream of the gene. These deletions are thought to remove enhancer element(s) required to activate SHOX limb expression. Because the SHOX gene and its surrounding sequences are very similar in all mammals except rodents, in this proposal we will use felines as model organisms to study SHOX gene regulation during limb development. The genomes of commonly used mammalian model organisms such as mice do not contain the SHOX gene, supporting the need for other, more pertinent models to explore the regulation of this gene. Therefore, we will use a technique called circular chromosome conformation capture (4C) to identify all sequences that interact with the feline SHOX gene during limb development. Such interacting sequences are excellent enhancer candidates. We will use the candidate sequences to identify the orthologous sequences in the human genome since we expect the regulation of the SHOX gene to be very similar in both species. The candidate sequences will then be analyzed for enhancer activity in cell culture assays. Identification of the enhancers regulating SHOX expression will be a key step in understanding the pathogenicity of genomic deletions linked to LWD and ISS phenotypes.